The goal of the proposed research is to understand the mechanisms by which eukaryotic cells regulate their metabolic pathways in response to changes in growth conditions. In the model system Saccharomyces cerevisiae, starvation for amino acids induces the GCN2 protein kinase to phosphorylate the alpha-subunit of eukaryotic translation initiation factor-2 (eIF-2). Phosphorylation of this factor stimulates the translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. Previously, it was shown that the sequence of the C-terminal region of GCN2 is homologous to the entire sequence of histidyl-tRNA synthetase (HisRS) from S. cerevisiae and humans. Given that aminoacyl-tRNA synthetases bind uncharged tRNA as a substrate and distinguish between charged and uncharged tRNA, it was proposed that the HisRS-related domain in GCN2 monitors the concentration of uncharged tRNA in the cell and activates the adjacent protein kinase moiety under starvation conditions when uncharged tRNA accumulates. A primary objective of this proposal is to test in a purified system whether the levels of uncharged tRNA regulate GCN2 phosphorylation of eIF-2-alpha. To test this model the following line of investigation is proposed. First, which amino acids are monitored in vivo by GCN2. It is known that GCN4 translation is stimulated in response to starvation for any one of at least ten different amino acids. Is GCN2 required for each limitation? Secondly, the interaction between different species of uncharged tRNA and GCN2 protein kinase will be measured by in vitro binding assays and the tRNAs shown to complex with GCN2 will be examined further to determine if they kinetically enhance the phosphorylation of eIF-2-alpha by GCN2. The final proposed experiments use genetic tools available in this system to isolate and characterize mutations in GCN2 with altered substrate specificity. A better understanding of the regulation and substrate affinity of GCN2 will provide useful information about stress responses in mammalian cells. It is known that eIF-2-alpha kinases related to GCN2 inhibit protein synthesis in response to viral infection (double-stranded RNA leads to activation of DAI kinase) and heme deprivation in reticulocytes (activates HCR protein kinase).